The long-term objectives of this proposal are to understand the process of evolution in asexual micro-organism populations, and their genetic structure. The wealth of genetic and biochemical information available for Escherichia coli and Saccharomyces cerevisiae permits a level of analysis not possible with many other organisms. Recent work has revealed that asexual micro-organism populations exhibit a wide-range of "interesting" and complex evolutionary responses that are analogous to evolutionary phenomena seen in diploid, sexually reproducing higher eukaryotes. In particular, populations initiated with a single clone, and grown in a simple unstructured environment, become polymorphic within a surprisingly short period of time. We will continue, and complete the analysis of a three component stable polymorphism that has been observed to develop in a population of E coli initiated with a single clone and grown in glucose-limited continuous culture for 765 generations. We will, i) determine the mechanisms responsible for interactions between the components, which serve to maintain the polymorphism, ii) determine the genetic bases for the interactions, by mapping, cloning and sequencing the loci responsible, iii) determine the necessary and sufficient conditions for the establishment of the polymorphism, and iv) determine the dynamics and fate of the polymorphism during continued evolution of the populations. In addition, we will analyze a unique "bank" of samples (taken every about 10 generations) from more that 40 evolving populations of E coli and S cerevisiae, to determine the genetic and biochemical bases for mechanisms which may result in the establishment of other stable polymorphisms. Here, our goal will be to determine the range of evolutionary changes which can generate and maintain genetic variation in simple, unstructured environments. As well as being important to population genetics and evolution, these studies will provide important information on the evolution and aging of proliferating cell populations. The complete understanding of cell proliferation and the genetic changes that cells undergo is critically important to many aspects of medicine, not the least of which are cancer therapy and aging.